A Novel 3-Dimentional and Multi-color Imaging by a Confocal Laser Microscopy Combined with Spectral Analyzer

Abstract

Various types of chromosomal abnormalities occur in tumor cells. Chromosomal translocation is one of the most popular abnormalities in hematopoietic tumor or many other tumors. It is known that specific chromosomal translocations, e.g. t(9,22) in chronic myeloid leukemia or t(11,22) in Ewing sarcoma, occur frequently and generate chimeric gene products providing oncogenic stimuli. Although the juxtaposition of two loci of genes in the nucleus is recently suggested to be an important factor for the cancer type-specific translocations, the mechanism have not been elucidated yet. If the three-dimensional relative positions among many kinds of genes or chromosome domains are simultaneously identified in the interphase nuclei, the important parts of the mechanism of chromosomal translocation can be discovered. In order to demonstrate this approach,the multi-color and 3-dimentional fluorescence microscopic system is required. However, only several different fluorescent dyes can be detected simultaneously by the conventional confocal laser microscopy because of overlapping fluorescent spectra. Therefore, we developed a novel imaging system composed of the nipkow-disk confocal scanner and spectral analyzer, that enables multi-color FISH analysis on the optical sections of the interphase nuclei. In this study, we focused on confirming the possibility to observe the optically sectioned images of various fluorescent specimens by way of our 3-D spectral analyzing technology.